The overriding aim of this proposal is to investigate the cellular and molecular mechanisms of vascular neotissue formation in tissue engineered vascular grafts (TEVG), with special emphasis placed on the mechanisms contributing to post-implantation TEVG stenosis. We have designed and developed a TEVG specifically for use in congenital heart surgery. Our TEVG is created by seeding autologous bone marrow derived-mononuclear cells (BM-MNC) onto a biodegradable tubular scaffold and briefly incubating the seeded scaffolds in autologous serum prior to implantation as a vascular conduit. We have performed the first clinical trial evaluating the use of our TEVG as a conduit connecting the inferior vena cava to the pulmonary artery in children requiring modified Fontan surgery. This pilot study demonstrated that our method is both safe and effective. This study also demonstrated that stenosis is the primary mode of graft failure. The rational design of improved "second-generation" vascular grafts will be predicated on our understanding of the mechanisms underlying TEVG stenosis. This research is based upon our preliminary data obtained using a murine model that faithfully recapitulates human vascular neotissue formation and the development of TEVG stenosis as exhibited in our human clinical trial. We hypothesize that BM-MNC seeded onto a tubular biodegradable scaffold and implanted as a vascular interposition graft produce monocyte chemoattractant protein-1 (MCP-1), which recruits circulating monocytes to the implanted TEVG. The monocytes differentiate into macrophages and infiltrate the graft. Subsequently, these macrophages recruit smooth muscle cells from the adjacent vessel wall through a platelet derived growth factor (PDGF)-dependent mechanism and endothelial cells via a vascular endothelial growth factor (VEGF)-dependent mechanism. Alterations in these processes can be used to modulate neotissue formation and to either promote or inhibit the formation stenosis. We will use transgenic mouse models to test these hypotheses in order to investigate the following specific aims: Aim 1: Determine if circulating monocytes are the cellular targets of MCP-1/CCR2 signaling and if the resulting macrophage infiltrate in TEVG is critical to the formation of vascular neotissue and development of TEVG stenosis. Aim 2: Determine the source of the smooth muscle cells that form the medial layer of the TEVG;determine the role of macrophage production of PDGF-B in mediating this process;and determine if modulation of macrophage production of PDGF will affect the formation of TEVG stenosis. Aim 3: Determine the source and identity of the cells that form the intimal layer of the neovessel;determine the role of macrophage production of VEGF-A in mediating this process;and determine if modulation of macrophage production of VEGF-A will affect TEVG stenosis. PUBLIC HEALTH RELEVANCE: Congenital cardiac anomalies are the most common birth defect and a leading cause of death in the newborn period. The most effective treatment for congenital cardiac anomalies is reconstructive surgery. Unfortunately, complications arising from the use of currently available vascular conduits are a significant cause of postoperative morbidity and mortality. The development of a tissue engineered vascular graft, created from an individual's own cells, with the ability to grow, repair, and remodel, holds great promise for advancing the field of congenital heart surgery and improving the outcomes of infants requiring surgical intervention.